Structural Biochemistry/Chromatography/Column

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Column Chromatography

Column chromatography is another method used to separate proteins or molecules from each other. It is essentially an upside-down version of TLC (Thin Layer Chromatography) - relying on the same physical principles, except that while TLC is driven by capillary forces for moving the solvent, column chromatography allows gravity to drive down the eluent. In this method, a sample to be separated is applied to the top of a glass column. the glass column is then packed with a solid phase. The purpose of this solid phase is to separate the compounds in the sample into different zones. Silica gel (SiO2) and alumina (Al2O3) are common adsorbents. The one expected to percolate out of the column first is the component that has the least interactions with the silica gel, so therefore the one that is least polar. The eluent carries the soluble compounds with it. When the column is packed with Silica Gel, the band expected to percolate out of the column first is the component that has the least interactions with the silica gel, so therefore the one that is least polar. The eluent carries the soluble compounds with it. The polarity of the eluent can be progressively increased from a nonpolar solvent to a polar solvent because as the nonpolar component is collected first, the bands of components left in the column are more polar. A more polar solvent would be more efficient to carry the polar component left in the column. After column chromatography is used to separate the mixture according to their respective polarities, Thin Layer chromatography should be used to separate the mixtures to observe the fractions separation and combine any that have “climbed” the same distance. The compounds found to have the same polarities using TLC are combined and analyzed by taking their respective melting points and comparing them to literature values. The amount of sample is used to figure out the initial concentration of each in the original sample. If the compound is colored this is easy, however if the compound is a clear solution then the plates can be CAM stained (Iodine can also be used) or put under UV to track their location. After these bands are collected, the solution can be put under a rotovap to evaporate off the solvent and a clean compound can be obtained. If the solvent is volatile, it can be evaporated in the hood or over night. The samples can also be heated in a sand bath.

Once the column is packed with dry stationary adsorbent material (such as silica gel), there are generally two methods to load column chromatography: wet loading and dry loading. The ability of the mixture to dissolve in a polar or non polar solvent determines the method of the column chromatography.

dry Column Chromatography separation
  • In wet loading method, the adsorbent is suspended in solvent and the slurry is transferred into the column as the eluent. This method is most commonly used when the desired separating mixture is soluble in the least polar solvent or a non-polar solvent. If excessively polar solvent is used, then it will stay inside the column and increase local polarity, which can mix the separation on the column.
  • In dry loading method, mixture is first dissolved in a minimal amount of solvent and the adsorbent material. Once the solvent mixed with the mixture and adsorbent is evaporated, the dried compound can be added into the column. After the addition of the dried compound, the column is flushed with mobile phase (can be polar solvent with various polarity, but they should be added with increasing polarity), and the column is not allowed to run dry after the addition of mobile phase. This method is most commonly used when the mixture is only soluble in solvent that are more polar than the eluent of choice.

The chemical compounds are separated and collected within the column. The separated sample can then be tested for purity and other properties. As the sample is applied to the top of the column, it is also washed with a solvent. As the sample moves over the solid phase in the column, the different molecules or compounds in the sample will begin to separate from each other into zones. The compounds in the sample will bind to the solid phase, but then the sample will also release from the solid phase and then bind to the liquid solvent that passes over it. This is a continuous process. A compound will bind to the solid phase, then release and bind to the solvent. it will then rebind back to the solid phase, and again rebind to the liquid solvent. This process keeps occurring as the compound moves down the column. Different molecules in the sample will have a different binding affinity to the solid phase or the liquid phase, these differences in affinity is what allows the molecule in a mixture to travel at different speeds and separate from the other compounds.(NOTE :*The preceding section was a description of column chromatography for organic chemistry.) This method is sometimes called reverse phase chromatography in biochemistry.

The factors that determine the distance a compound travels are 1) the interaction between the solvent and adsorption layer, 2) the interaction between the solute and adsorption layer, 3) the polarity of the solute, solvent, and adsorption layer, and 4) the weight of the solution. To determine the distance a compound travels one may calculate the retardation factor (Rf). The Rf value can be found by looking at a TLC plate that is spotted with the fractions collected after running a column. The Rf value is the ratio of the distance traveled by the solute over the distance traveled by the solvent. The range of Rf is 0 to 1. If the calculated Rf value is higher than desired, then a less polar solvent should be used when running the column.


Organic Chemistry Laboratory third edition with Qualitative Analysis,By Bell Jr, Charles E; Taber, Douglas F.; Clark, Allen K. Harcourt College Publisher